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PubMed1983

Inherited complement component deficiencies in membranoproliferative glomerulonephritis.

Anecdotal reports of complement component deficiencies in patients with immune complex disease led to a systematic study of the levels of seven complement components in serum specimens from 178 patients with glomerulonephritis and 163 normal subjects. Deficiencies were found with significantly higher frequency (22.7%) among 44 patients with membranoproliferative glomerulonephritis (MPGN) types I and III, than among the normal subjects (6.7%, P less than 0.002) or among 134 patients with other glomerulonephritides (5.2%, P less than 0.001). The component deficiencies in MPGN were partial in nine patients and subtotal in one. They could not be ascribed to acquired hypocomplementemia or to a nephrotic syndrome. They were present over long periods, were found in family members, and involved C2, C3, factor B, C6, C7, and C8. Six were presumably the result of null structural genes, two were associated with a structurally abnormal component, and two were of unknown cause. The results give evidence that partial deficiency of one or more complement components is a factor predisposing to MPGN.

Adolescent↗
PubMed1999

Peritoneal mesothelial cells produce complement factors and express CD59 that inhibits C5b-9-mediated cell lysis.

The CD59 membrane protein confers protection from C5b-9-mediated cell lysis. Because evidence exists for complement (C) activation and generation of C5b-9 in the peritoneal cavity during chronic peritoneal dialysis (CPD), we investigated, on mesothelial cell (MC) lines, the expression of CD59 and the production of C components. Four MC lines were obtained from children on CPD, and two from non uremic children. CD59 expression on MCs was investigated with anti-CD59 monoclonal antibody (mAb) and polyclonal goat immunoglobulin G (IgG). MC lines were positive for staining with anti-CD59 mAb. Western blotting analysis of MC membrane demonstrated a band with the same molecular weight as CD59. Incubation of MC with anti-CD59 mAb abrogated the protective effect of CD59 (100% cytotoxicity). C3, C4, and C6 were detected in the supernatants of MC; in non uremic MC supernatants, C5, C7, C8, and C9 were also detectable, and C4 concentration was tenfold higher. CD59 expression confers to MCs protection from C5b-9-mediated lysis. MCs produce C factors. These findings suggest that production of complement components and expression of CD59 on MCs could play a role both in peritoneal cavity infection (decreased complement production) and in peritoneal membrane damage (decreased CD59 expression and reduced remesothelialization owing to MC lysis).

CD59 Antigens↗
PubMed1999

Complement regulators C1 inhibitor and CD59 do not significantly inhibit complement activation in Alzheimer disease.

Proteins characteristic of activated complement are associated with Alzheimer disease (AD) lesions. The classical complement pathway can be activated only when the influence of such endogenous regulators as C1-inhibitor (C1-inh) and CD59 are overcome. We used the techniques of reverse transcriptase-polymerase chain reaction and Western blotting to assess the mRNA and protein levels of C1-inh and CD59 in AD and control brains in comparison with levels of the complement components with which they interact. The inhibitors were only slightly upregulated and then only in heavily affected areas of AD brain such as the entorhinal cortex, hippocampus, midtemporal gyrus and midfrontal gyrus. The ratio of AD to control mRNAs in these four areas was 1.17 for C1-inh and 1.12 for CD59, compared to 3.06 for C1r, 2.67 for C1s, 2.35 for C5, 2.56 for C6, 2.42 for C7, 5. 08 for C8 and 16.3 for C9. Peripheral organ expression of C1-inh and CD59 mRNAs was no different in AD than controls but was slightly upregulated in infarcted heart tissue. Again, the increase was small compared with that of the competitive complement components. These data indicate that the forces which upregulate and activate complement in AD and myocardial infarction are not effectively suppressed by the endogenous regulators, C1-inh and CD59.

Adult↗
PubMed2000

Identification of mutations in rat CD59 that increase the complement regulatory activity.

Formation of the membrane attack complex (MAC) of complement on host cells is inhibited by the glycosylphosphatidylinositol- (GPI-) anchored glycoprotein CD59. Published data on the active site of human CD59 are confusing. To clarify these data, we set out to elucidate the active site of a nonprimate CD59 molecule by site-directed mutagenesis. We also undertook to investigate a region of potential species selectivity, and to this end rat CD59 was chosen for all mutations. Our investigations confirmed the proposal that the active site of CD59 is the major hydrophobic groove, with mutations Y36A, W40A, and L54A ablating complement inhibitory function of CD59. Other mutations reducing the function of rat CD59 were I56E, D24A, and D24R. Importantly, mutations at one residue increased the function of rat CD59. The K48E mutation significantly increased function against human rat or rabbit serum, whereas the K48A mutation increased function against human serum alone. A similar mutation in human CD59 (N48E) had no effect on activity against human or rat serum but completely abolished all activity against rabbit serum. These findings suggest that the alpha-helix of human CD59, adjacent to the hydrophobic groove, influences the interaction between human CD59 and rabbit C8, C9, or both.

Amino Acid Substitution↗
PubMed1996

Complement activation by myeloperoxidase products released from stimulated human polymorphonuclear leukocytes.

Purified human myeloperoxidase (MPO) converted human C5 to an activated form, i.e. the C5 protein adopted a configuration expressing a binding site for C6; the resulting C56 complex then reacted with C7, C8 and C9 forming a hemolytic C5-9 complex. For the activation by myeloperoxidase chloride and hydrogen peroxide were essential. This indicates that the peroxidase acted through the generation of HOCl which had been shown earlier to oxidize and activate C5. Human polymorphonuclear leukocytes (PMN) were stimulated in vitro by incubation with opsonized zymosan; thereafter the supernatants were tested for C5 activating potency. Stimulated PMN release H2O2 and MPO that produces hypochlorite and secondarily various chloramines. As a trap for the labile hypochlorite generated excess taurine was added to the PMN suspensions during the incubation. Hypochlorite is then stoichiometrically converted to the relatively stable taurine chloramine. In order to rule out interfering activities of proteolytic enzymes released from the PMN and known to attack C5, the supernatants were ultracentrifuged, and the ultrafiltrates, containing only low molecular weight compounds, were used for the further studies. They contained taurine chloramine, estimated photometrically, and they activated C5 upon incubation, assayed functionally by reactive lysis. Azide, an inhibitor of myeloperoxidase, and catalase which destroys H2O2, essential for MPO-catalyzed oxidations, prevented the generation of C5 activating potency and of chloramines. Unstimulated PMN produced neither oxidants nor C5 activating potency. When taurine was omitted from the PMN suspensions during stimulations much less oxidant was found in the supernatants and less C5 activating potency. These findings indicate that the C5 activating agent was produced by stimulated PMN through MPO-generated hypochlorite, trapped as taurine chloramine. In the absence of added taurine the hypochlorite formed by MPO oxidized endogenous amines that also activated C5. Further studies suggested that among these was some monochloramine derived from endogenous ammonia. Activation of the terminal complement reaction sequence by MPO released from stimulated PMN may represent a third pathway to complement activation contributing to and reinforcing complement and PMN functions at the site of inflammation or tissue injury.

Complement Activation↗
PubMedDate not supplied

Domain structure, functional activity, and polymerization of trout complement protein C9.

The 3' region of trout C9 has been resequenced and found to differ from the previously published sequence (Stanley and Herz, EMBO J. 6:1951; 1987). In contrast to other sequenced C9 molecules, but in common with the other terminal complement components, trout C9 was found to contain an additional carboxy terminal thrombospondin domain. This domain does not restrict polymerization, as has been previously suggested (Stanley and Luzio, Nature 334:475; 1988), since alternative pathway activation of trout complement by rabbit erythrocytes lead to the formation of circular membrane attack complement lesions on the erythrocyte membrane. Although the trout C9 molecule is larger than human C9, the diameters of circular trout membrane attack complexes were approximately 30% smaller than their human counterparts. No lysis of erythrocytes bearing human C5b-7 or C5b-8 complexes was detected following incubation with trout serum containing EDTA, which suggests that trout C8 and C9 are unable to bind to human C7 and C8, respectively. Finally, trout and human serum were equally effective at killing the human serum-sensitive strain Salmonella minnesota Re595.

Amino Acid Sequence↗
PubMed1986

C5b-9 assembly: average binding of one C9 molecule to C5b-8 without poly-C9 formation generates a stable transmembrane pore.

Membrane attack by serum complement normally results in the formation of C5b-9 complexes that are heterogeneous with respect to their C9 content. We here report that an apparently homogeneous population of C5b-9 complexes can be generated through treatment of C5b-7-laden sheep erythrocytes with C8 and C9 for 60 min at 0 degree C. Experiments performed by using radioiodinated C8 and C9 components have indicated that binding of C8 to these target cells is essentially temperature independent. In contrast, when a surplus of C9 molecules is offered to C5b-8 cells, an approximately fourfold to 4.5-fold higher number of C9 molecules become cell bound at 37 degrees C as opposed to 0 degree C. C5b-9 complexes isolated from target membranes treated with C9 at 0 degree C contain no polymerized C9 and do not exhibit the ring structure characteristic of the classical complement lesion. Nevertheless, these complexes generate stable transmembrane channels and cause hemolysis at 37 degrees C. The pores have been sized to 1 to 3 nm effective diameter by osmotic protection experiments. SDS-PAGE of the isolated complexes indicates an average stoichiometry of only one molecule C9 bound per C5b-8 complex. The results show that oligomerization of C9 with formation of ring lesions is not a basic requirement for the generation of stable transmembrane complement pores in sheep erythrocytes. They indirectly support the contention that terminal complement components other than C9 contribute to the intramembrane domains of C5b-9 pores.

Animals↗
PubMed1997

Presence of immunoglobulins, C3 and cytolytic C5b-9 complement components on the surface of erythrocytes from patients with beta-thalassaemia/HbE disease.

The occurrence of IgG, IgM, IgA, C3 and C5b-9 complement complexes on erythrocytes from 43 patients with beta-thalassaemia HbE disease was investigated. Indirect immunoradiometric assays using radioiodinated protein A were employed to quantify the individual components. We confirmed that circulating erythrocytes from thalassaemic patients contained elevated amounts of IgG, and small but significant amounts of C3. In addition, small but significant amounts of C5b-9 were detected. Levels of cell-bound IgG, C3 and C5b-9 were higher in splenectomized versus non-splenectomized patients. The presence of C5b-9 on circulating cells from five splenectomized patients was confirmed by an ELISA employing a monoclonal antibody specific for a C5b-9 neoantigen. When C5b-9 positive cells from two patients were solubilized with detergent and subjected to sucrose density gradient centrifugation, the terminal complexes sedimented as 25-40S macromolecules, thus behaving as membrane C5b-9 complexes. The presence of C8 and C9 in these high molecular weight fractions was directly demonstrated by Western blotting. These results constitute the first demonstration that circulating diseased erythrocytes may carry low numbers of potentially cytolytic C5b-9 complement complexes which may be partly responsible for the known ionic disturbances found in thalassaemic cells. Both bound C3 and C5b-9 could promote removal of diseased cells in the reticuloendothelial system.

Adult↗
PubMed1993

Activation of the alternative complement pathway and production of factor H by skeletal myotubes.

Skeletal muscle myotubes from neonatal rats were used to study the interaction of skeletal muscle with complement. Serum from guinea pig, rabbit, and human, in the absence of muscle-specific antibody, caused creatine phosphokinase release, which required activation of the terminal complement cascade. Cleavage of serum C3 and Factor B in the presence of myotubes was dependent on Mg2+, but not Ca2+, and C3 cleavage occurred only in the presence of Factor B. Rat myotubes caused significant consumption of C8 and C9 in rat serum, which also required Mg2+, but not Ca2+. All of these findings are typical of a tissue capable of activating the alternative pathway. In addition, the C2 myotube cell line was shown to produce Factor H, an inhibitory protein of the alternative pathway, as demonstrated by Factor H mRNA expression and immunoprecipitation of the protein.

Animals↗
PubMed1992

Complement and pregnancy: new insights into the immunobiology of the fetomaternal relationship.

Recent studies have revealed that human trophoblast expresses three membrane-bound proteins which function specifically to regulate the activity of complement. These proteins are already known to be widely distributed in normal adult tissues where they protect host cells from damage resulting from the fortuitous deposition of activated complement components. Their activities are focused at two distinct steps in the complement pathway. Decay accelerating factor (DAF, CD55) and membrane co-factor protein (MCP, CD46) act at the level of the C3 convertase enzymes which activate C3 to C3b. A further protein, CD59, directly regulates the formation and function of the terminal cytolytic membrane attack complex (MAC) by specifically interacting with C8 and C9. These proteins appear to play an important role in the maintenance of normal human pregnancy. DAF, MCP and CD59 are all expressed where trophoblast surfaces are in contact with maternal blood and tissues and expression occurs from at least 6 weeks of gestation. The semi-allogeneic human conceptus therefore appears to be effectively protected from maternal complement-mediated damage arising either from alternative or classical pathway activation or in a bystander fashion following a response to microbial infection in the mother. Complement regulatory protein deficiency disorders with clinically demonstrable consequences especially in terms of haemolytic disease are known to exist and have proved valuable in establishing the biological role of these proteins in vivo. The demonstration of this new family of immunoregulatory proteins on trophoblast raises important questions about the potential involvement of these products in pregnancy pathologies.

Complement C3↗
PubMed1986

The membrane attack complex in complement-mediated glomerular epithelial cell injury: formation and stability of C5b-9 and C5b-7 in rat membranous nephropathy.

Using a model of rat membranous nephropathy (MN), we examined the relationship between the development of glomerular epithelial cell injury and the formation and stability of the membrane attack complex (MAC) of complement. Isolated rat kidneys were perfused with buffered bovine albumin (BSA) or various plasmas (complement source). Kidneys containing nephritogenic amounts of complement-fixing sheep antibody to glomerular epithelial antigens (aFx1A) perfused with BSA (n = 5), and normal kidneys perfused with normal human plasma in BSA (50% v/v, n = 6) excreted 0.30 +/- 0.02 mg protein/min/g during 90 min perfusion (control groups). When normal plasma was added to the perfusate of aFx1A kidneys at concentrations of 12.5, 25, and 50% v/v, protein excretion rose in a time- and concentration-dependent manner. Perfusions with 25% plasma resulted in baseline proteinuria from 0 to 20 min that increased to 2.8 +/- 0.9 mg/min/g at 20 to 40 min and 8.6 +/- 2.1 at 40 to 60 min (n = 4, p less than 0.01 vs control groups). Removal of plasma at 20 min did not prevent this rise in protein excretion (3.9 +/- 2.4 and 5.8 +/- 2.6 mg/min/g at 30 to 40 and 55 to 65 min respectively, p less than 0.01, n = 4). Perfusion of aFx1A kidneys with C8-deficient (C8D) human plasma (25% v/v, n = 4) or C6D rabbit serum (25% v/v, n = 2) independently produced low levels of proteinuria comparable with BSA, but in combination, the two reagents restored enhanced protein excretion (n = 2). In aFx1A kidneys containing C5b-7, addition of C8 and C9 (C6D serum) after intervals of 20, 60, or 90 min immediately reconstituted heavy proteinuria. Thus, the magnitude of MAC-induced glomerular epithelial injury in rat MN is related to the complement dose. Altered glomerular permeability is delayed with respect to the onset of complement activation. Once sufficient C5b-9 is formed, proteinuria can develop despite cessation of new MAC assembly, implying that C5b-9 persists after formation. Moreover, the C5b-7 MAC intermediate is not eliminated rapidly in this model.

Animals↗
PubMed1994

Identity of a peptide domain of human C9 that is bound by the cell-surface complement inhibitor, CD59.

The CD59 antigen is a plasma membrane glycoprotein that serves as an inhibitor of the C5b-9 complex of complement. This inhibitory activity appears related to the capacity of CD59 to bind with high affinity to sites that are nascently exposed in the alpha-chain subunit of human C8, as well as within the C9b domain (amino acid residues 245-538) of human C9, during assembly of the C5b-9 complex on the target membrane (Ninomiya, H., and Sims, P. J. (1992) J. Biol. Chem. 267, 13675-13680). The CD59 binding site in C9 was first investigated by N-terminal sequencing of CD59-binding peptides generated by limited digest of the isolated C9b domain. These experiments revealed a 17-kDa fragment (starting at C9 residue Thr-320) that retained affinity for CD59, suggesting the possibility for localizing the CD59 binding site by mapping with small C9-derived peptides. Peptides spanning the entire C9b sequence were expressed in Escherichia coli and then probed with CD59. CD59 bound specifically to all peptides starting N-terminal to C9 residue 359 with C termini extending beyond residue 411. Little to no CD59 binding was observed for various C9-derived peptides that started C-terminal to residue 359 or that were truncated N-terminal to residue 411. Affinity-purified antibody against C9 residues 320-411 inhibited CD59 binding to C9 by > 50% and completely inhibited its binding to the isolated C9b domain. Little to no specific binding of CD59 was detected for peptides restricted to the putative hinge domain within C9b (residues 245-271). These results indicate that a CD59 binding site is located between residues 320 and 411 of the C9 polypeptide and suggest that the affinity of this site is principally determined by residues 359-411.

Amino Acid Sequence↗
PubMed1991

Nuclease S1-mediated enhancement of the 32P-postlabeling assay for aromatic carcinogen-DNA adducts.

Treatment of DNA digests with nuclease P1 prior to 32P-labeling of adducts has previously been shown to enhance the sensitivity of the 32P-postlabeling assay for the detection of aromatic carcinogen-DNA adducts. The enhancement was based on the ability of nuclease P1 to remove the 3'-phosphate from normal nucleotides but not the corresponding phosphate from most aromatic adducted nucleotides. We investigated the utility of another 3'-dephosphorylating enzyme, nuclease S1, for this purpose, and found it to be as effective as nuclease P1. The recovery of DNA adducts derived from benzo[a]-pyrene (B[a]P), benzoquinone (BQ) and 2-acetylaminofluorene (AAF) was comparable after enhancement with either enzyme. Some differences were, however, observed. Recovery of a minor B[a]P adduct was 1.5 times higher by the S1 procedure. Among minor adducts of BQ, two showed higher values (2.8- and 6.1-fold) by the S1 procedure and one by the P1 procedure (2.4-fold). The major AAF adduct, deoxyguanosine-C8-AF, exhibited poorer recovery (1-11%) by either procedure, while the minor adducts, deoxyguanosine-N2-AAF and deoxyguanosine-C8-AAF, showed better recovery (2-3 times) than by the enhancement procedure involving extraction of adducts into butanol. Our results show that the nuclease S1 assay can complement the nuclease P1 assay, with improved recoveries for some adducts. Considering the complexity of the postlabeling assay, this additional variant may prove useful in unequivocal detection of DNA adducts.

2-Acetylaminofluorene↗
PubMed1986

In vitro inhibition of the classical pathway of human complement by a natural microbial product, colistin sulphate.

Colistin sulphate was found to be an inhibitor of the classical pathway of the complement system. The main sites of inhibition were the interaction of EAC14 with C2 and EAC142 with C3. It also inhibited EAC14 formation from EA and C2-deficient serum, EAC1-7 formation from EAC1-3, C5, C6 and C7 and the interaction of EAC1-7 with C8 and C9, though less efficiently. It did not inhibit formation of C3/C5 convertase of the alternative pathway. The inhibition of the classical pathway was reversible since hemolytic activity was completely restored after dialysis.

Animals↗
PubMed1978

Assembly of the cytolytic alternative pathway of complement from 11 isolated plasma proteins.

The known cytolytic function of the alternative pathway in serum was quantitatively reproduced by combining 11 isolated plasma proteins at their respective serum concentrations. These proteins are: C3, Factor B, Factor D, C3b inactivator, beta1H, native properdin, C5, C6, C7, C8, and C9. In absence of activators of the alternative pathway, this mixture was stable at 37 degrees C as evidenced by lack of consumption of Factor B, C3, and C5. Upon addition of either rabbit erythrocytes or neuraminidase-treated sheep erythrocytes, cell lysis ensued and the extent of lysis was dependent on dose of the component mixture. The dose-response curves obtained with the isolated component mixture and with C4-depleted serum were virtually indistinguishable. Nonactivator erythrocytes (untreated sheep erythrocytes) were not lysed by the component mixture. Deletion of properdin resulted only in a twofold diminution of the hemolytic activity of the component mixture. No immunoglobulin requirement was apparent. These results indicate that the cytolytic systems studied are internally sufficient and capable of coupling the initiation and amplification sequence with the cytolytic membrane attack sequence.

Cell-Free System↗
PubMed1987

Formation of transmural complement pores in serum-sensitive Escherichia coli.

The binding of C9 at 0 and 37 degrees C to viable Escherichia coli K-12 cells carrying C5b-8 complexes was quantified. At low temperature, limited average binding of only 1 to 1.4 molecules of C9 per C8 molecule occurred, whereas 6 to 8 C9 molecules were bound per C8 molecule at 37 degrees C. Despite incorporation of C9 into C5b-9 complexes at 0 degrees C, these terminal complexes caused no loss of bacterial viability even when present in very large numbers (1,000 to 1,500 per CFU) on the bacterial cells. In contrast, generation of 50 to 100 C5b-9 complexes carrying multiple C9 molecules per CFU caused loss of viability. The failure of C5b-81C91 complexes to generate transmural pores was confirmed by measurements of o-nitrophenyl-beta-D-galactoside influx into the cells. Whereas treatment of C5b-8-laden cells with C9 at 32 degrees C caused virtually instantaneous influx of the marker, almost no influx was registered in cells receiving C9 at 0 degrees C. When cells carrying C5b-7 were brought into the stationary phase and given C8 and C9 at 32 degrees C, a C9-dependent disruption of the outer membrane permeability barrier immediately occurred as demonstrated by cleavage of a chromogenic substrate by periplasmic beta-lactamase. In sharp contrast, o-nitrophenyl-beta-D-galactoside influx was markedly retarded over a prolonged period, with abrupt permeability increases of the inner membrane toward this molecule being noted just before bacterial cell division occurred. We conclude that killing of E. coli requires binding of C5b-9 complexes containing C9 oligomers to the outer membrane and suggest that formation of pores in the inner membrane occurs when these complexes are "hit" by transiently forming zones of bioadhesion. Formation of the latter may be a dynamic process that is accentuated during cell division and quiescent during the stationary phase.

Blood Bactericidal Activity↗
PubMed1992

Contribution of the N-linked carbohydrate of erythrocyte antigen CD59 to its complement-inhibitory activity.

The contribution of N-linked carbohydrate to the complement-inhibitory function of the human erythrocyte membrane glycoprotein, CD59, was investigated. Amino acid sequence analysis of tryptic peptides labeled with [3H]borohydride revealed an N-linked carbohydrate moiety at the Asn18 residue. No O-linked carbohydrate was detected, as judged by the failure of asialo-CD59 to bind peanut agglutinin and by its resistance to digestion by O-glycanase. The apparent molecular mass of CD59 was reduced from 18-20 to 14 kDa upon complete digestion with N-glycanase, with no detectable proteolysis. N-glycanase digestion of CD59 was associated with an 88 +/- 4% loss of the complement-inhibitory activity of the protein, as assessed by its capacity to protect chicken erythrocytes from lysis by the human C5b-9 proteins. By contrast, no change in function was observed after digestion of CD59 with neuraminidase, under conditions that removed greater than 60% of [3H]sialic acid residues. Despite loss of functional activity after N-glycanase digestion, we detected no change in the capacity of the deglycosylated CD59 to incorporate into erythrocyte membranes or to bind specifically and with species selectivity to the C8 and C9 components of the membrane attack complex. In order to alter the branched-chain structure of the N-linked carbohydrate of CD59 without enzymatic digestion, Chinese hamster ovary (CHO) cells transfected with cDNA for human CD59 were grown in the alpha-mannosidase inhibitor, 1-deoxymannojirimycin, resulting in conversion of approximately 70% of the membrane glycoprotein to a high mannose. When grown in the presence of 1-deoxymannojirimycin, the C5b-9-inhibitory activity of CD59 expressed on the surface of the transfected CHO cells was reduced by an amount comparable to that observed for the N-glycanase digested protein. Taken together, these data suggest that normal glycosylation of Asn18 in CD59 is required for the normal expression of its complement-inhibitory activity on membrane surfaces, although these N-linked sugar residues do not contribute to CD59's affinity for the C8 and C9 components of the C5b-9 complex.

Animals↗